Field of the Invention
The present invention relates to a method for secretory production of a heterologous protein.
Description of the Related Art
Methods for secretory production of heterologous proteins by microorganisms have been reported in, for example, a Bacillus bacterium (Microbiol. Rev., 57, 109-137 (1993)), methanol-assimilating yeast, Pichia pastoris (Biotechnol., 11, 905-910 (1993)), filamentous fungi of the genus Aspergillus (Biotechnol., 6, 1419-1422 (1988); Biotechnol., 9, 976-981 (1991)), and so forth.
Secretory production of heterologous proteins in coryneform bacteria has also been attempted, and methods reported include secretion of a nuclease and a lipase by Corynebacterium glutamicum (henceforth also abbreviated as C. glutamicum) (U.S. Pat. No. 4,965,197, J. Bacteriol., 174, 1854-1861 (1992)), secretion of a protease such as subtilisin (Appl. Environ. Microbiol., 61, 1610-1613 (1995)), secretion of a protein using signal peptides of cell surface layer proteins PS1 and PS2 (also referred to as CspB) of coryneform bacteria (Japanese Patent Laid-open (Kohyo) No. 6-502548), secretion of a fibronectin-binding protein using the signal peptide of PS2 (CspB) (Appl. Environ. Microbiol., 61, 1610-1613 (1995)), secretion of protransglutaminase using signal peptides of PS2 (CspB) and SlpA (also referred to as CspA) (Japanese Patent No. 4320769), secretion of a protein using a variant type secretion system (Japanese Patent Laid-open (Kokai) No. 11-169182), secretion of a protransglutaminase by a variant strain (Japanese Patent No. 4362651), and so forth. In addition, techniques for improving secretory production amounts of heterologous proteins by coryneform bacteria are known and include reducing the activity of a cell surface layer protein (WO2013/065869 and WO2013/065772), reducing the activity of a penicillin-binding protein (WO2013/065869), enhancing the expression of a gene encoding a metallopeptidase (WO2013/065772), introducing a mutation into a ribosomal protein S1 gene (WO2013/118544), expressing a heterologous protein with an amino acid sequence comprising Gln-Glu-Thr inserted between a signal peptide and the heterologous protein (WO2013/062029), and so forth.
The “Sec system” is a general protein secretion pathway and is widely present in both prokaryotes and eukaryotes; however, an entirely different protein secretion pathway has recently been found in thylakoid membranes of chloroplasts of plant cells (EMBO J., 14, 2715-2722(1995)). This novel secretory pathway has been named the “Tat system” (Twin-Arginine Translocation system) due to the arginine-arginine sequence that is commonly present in the signal sequence of a secreted protein secreted (EMBO J., 14, 2715-2722(1995)). Proteins secreted by the Sec system are in a precursor state before forming a higher-order structure, while proteins secreted by the Tat system through the cell membrane have already formed a higher-order structure in the cell (J. Biol. Chem., 25; 273(52), 34868-74(1998)). Secretory production of proteins utilizing a Tat-dependent signal peptide has been reported in coryneform bacteria (WO2013/118544; Japanese Patent No. 4730302).
A “two-component regulatory system” is a system in which bacteria respond to various environmental changes both inside and outside of the cell. The two-component regulatory system has two components: a sensor kinase that is responsible for sensing an environmental change stimulus, and a response regulator that is responsible for receiving a signal from the sensor kinase and regulating the expression of downstream genes. When the sensor kinase senses a stimulus, a specific histidine residue in the kinase is autophosphorylated, a signal is transduced via transfer of the phosphate group to a specific aspartic acid residue in the response regulator, and thereby the phosphorylated response regulator is activated as a transcription factor to regulate the expression of the downstream genes.
The two-component regulatory system of C. glutamicum is described in Appl. Microbiol. Biotechnol., 94, 1131-1150(2012), etc. For C. glutamicum, at least 13 types of systems are known to be two-component regulatory systems. One of these is the PhoRS system, which includes a sensor kinase PhoS protein and a response regulator PhoR protein. Analysis of a PhoRS-deficient strain revealed that the PhoRS system is a regulatory system that senses phosphate depletion in the environment and performs signal transduction (J. Bacteriol., 188, 724-732(2006)).
The PhoS protein is a membrane protein having two transmembrane domains. The PhoS protein includes a sensor domain that senses a stimulus, a linker domain called a HAMP domain, a HisKA domain having a histidine residue that is autophosphorylated, and a HATPase domain that is able to bind ATP catalyze autophosphorylation of the histidine residue. The PhoR protein is an intracellular protein that includes a receiver domain on the N-terminal side that receives a signal, and an effector domain on the C-terminal side that regulates the expression of the downstream genes (Appl. Microbiol. Biotechnol., 94, 1131-1150(2012)).
However, the relationship between the PhoRS system and the secretory production of heterologous proteins has not been previously described. Also, the effect of a mutation of the PhoS protein is unknown, nor has such a mutation been reported to be effective for secretory production of heterologous proteins in coryneform bacteria. Furthermore, that the effect of specific mutations of the PhoS protein and their relationship to secretory production of heterologous proteins has not been previously reported.